Asymmetric ester hydrolysis with pig-liver esterase

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Asymmetric ester hydrolysis with pig liver esterase is the enantioselective conversion of an ester to a carboxylic acid through the action of the enzyme pig liver esterase (EC 3.1.1.1). Asymmetric ester hydrolysis involves the selective reaction of one of a pair of either enantiotopic (within the same molecule and related by a symmetry plane of the molecule) or enantiomorphic (in enantiomeric molecules and related as mirror images) ester groups. [1]

Enantioselective synthesis

Enantioselective synthesis, also called asymmetric synthesis, is a form of chemical synthesis. It is defined by IUPAC as: a chemical reaction in which one or more new elements of chirality are formed in a substrate molecule and which produces the stereoisomeric products in unequal amounts.

Ester chemical compounds consisting of a carbonyl adjacent to an ether linkage

In chemistry, an ester is a chemical compound derived from an acid in which at least one –OH (hydroxyl) group is replaced by an –O–alkyl (alkoxy) group. Usually, esters are derived from a carboxylic acid and an alcohol. Glycerides, which are fatty acid esters of glycerol, are important esters in biology, being one of the main classes of lipids, and making up the bulk of animal fats and vegetable oils. Esters with low molecular weight are commonly used as fragrances and found in essential oils and pheromones. Phosphoesters form the backbone of DNA molecules. Nitrate esters, such as nitroglycerin, are known for their explosive properties, while polyesters are important plastics, with monomers linked by ester moieties. Esters usually have a sweet smell and are considered high-quality solvents for a broad array of plastics, plasticizers, resins, and lacquers. They are also one of the largest classes of synthetic lubricants on the commercial market.

Carboxylic acid oxoacid having the structure RC(=O)OH, used as a suffix in systematic name formation to denote the –C(=O)OH group including its carbon atom

A carboxylic acid is an organic compound that contains a carboxyl group. The general formula of a carboxylic acid is R–COOH, with R referring to the rest of the molecule. Carboxylic acids occur widely. Important examples include the amino acids and acetic acid. Deprotonation of a carboxyl group gives a carboxylate anion.

Contents

Introduction

Enzymes, which are composed of chiral amino acids, catalyze chemical reactions with high stereoselectivity. Specifically, esterase enzymes catalyze the hydrolysis of esters to carboxylic acids. This transformation may be rendered asymmetric if two enantiotopic ester groups exist in the substrate or if a racemic mixture of chiral esters is used. In the former case (desymmetrization), the chiral environment of the enzyme active site leads to selective hydrolysis of the ester that is closer to the catalytically active serine residue when the substrate is bound to the enzyme. In the latter case (kinetic resolution), one of the enantiomers is hydrolyzed faster than the other, leading to an excess of hydrolyzed product from one enantiomer. Both strategies rely on the fact that the transition states for hydrolysis of enantiotopic or enantiomorphic ester groups by the chiral enzyme are diastereomeric. [2]

In chemistry, stereoselectivity is the property of a chemical reaction in which a single reactant forms an unequal mixture of stereoisomers during a non-stereospecific creation of a new stereocenter or during a non-stereospecific transformation of a pre-existing one. The selectivity arises from differences in steric effects and electronic effects in the mechanistic pathways leading to the different products. Stereoselectivity can vary in degree but it can never be total since the activation energy difference between the two pathways is finite. Both products are at least possible and merely differ in amount. However, in favorable cases, the minor stereoisomer may not be detectable by the analytic methods used.

Asymmetry state; the absence of, or a violation of, symmetry

Asymmetry is the absence of, or a violation of, symmetry. Symmetry is an important property of both physical and abstract systems and it may be displayed in precise terms or in more aesthetic terms. The absence of or violation of symmetry that are either expected or desired can have important consequences for a system.

In chemistry, a racemic mixture, or racemate, is one that has equal amounts of left- and right-handed enantiomers of a chiral molecule. The first known racemic mixture was racemic acid, which Louis Pasteur found to be a mixture of the two enantiomeric isomers of tartaric acid. A sample with only a single enantiomer is an enantiomerically pure or enantiopure compound.

Pig liver esterase (PLE) is a widely used enzyme for asymmetric ester hydrolysis. Although it was originally used for the desymmetrizing hydrolysis of glutarate esters, [3] PLE also hydrolyzes malonates, cyclic diesters, monoesters, and other substrates. Active site models have been advanced to explain the selectivity of PLE. [4]

Malonate salts and esters of malonic acid

The malonate or propanedioate ion is CH
2(COO)2−
2 (malonic acid minus two hydrogen ions). Malonate compounds include salts and esters of malonic acid, such as

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EsteraseGen.png

Mechanism and Stereochemistry

Prevailing Mechanism

The active site of PLE facilitates both substrate binding and hydrolysis. A key serine residue in the active site promotes hydrolysis, but the substrate must present an ester group to this residue after binding to the enzyme active site for hydrolysis to take place. Whether the substrate is able to present an ester group to the catalytic serine residue depends on its bound conformation in the active site, which is dictated by amino acid side-chains in the active site. Thus, active site models of PLE have been advanced with the goal of predicting from the structure of the substrate which of two enantiotopic ester groups will be hydrolyzed (or whether hydrolysis is likely to occur at all).

A simple model for the binding conformation of an ester in the active site of PLE is shown below. This model accurately predicts the configuration of hydrolyzed glutarates and similar substrates.

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EsteraseMech.png

Scope and Limitations

Although the substrate scope of PLE is broad, enantioselectivity varies as a function of the structure of the substrate. This section describes substrates that are hydrolyzed by PLE with the highest enantioselectivity, as well as sensitive substrates that may be hydrolyzed to achiral carboxylic acids in high yield without side reactions.

Chirality property of an object that is distinguishable from its mirror image

Chirality is a property of asymmetry important in several branches of science. The word chirality is derived from the Greek χειρ (kheir), "hand," a familiar chiral object.

Glutarates were the first substrates to be hydrolyzed with PLE in high enantioselectivity. Although yields are moderate, enantioselectivity is extremely high. [5]

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3-Alkyl glutarates with small alkyl substituents are hydrolyzed to the (R)-monoester; however, when a large alkyl substituent is present, the (S)-monoester forms. [6] This switch in enantioselectivity is accurately predicted by the active site model given above.

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EsteraseScope2.png

An opposite trend is observed in desymmetrizing hydrolyses of 2-methyl malonates, which afford the (S) enantiomer when the other substituent on C-2 is small, and the (R) enantiomer when the other C-2 substituent is large. [7]

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EsteraseScope3.png

A number of meso diesters other than the substrates described above may be hydrolyzed by PLE with high enantioselectivity. Cyclic meso diesters tend to be hydrolyzed more selectively than acyclic diesters. [8] The predominant enantiomer of product depends on ring size. [9] [10]

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7-Oxabicyclo[2.2.1]heptane-2,3-dicarboxylates are an interesting class of diesters that are hydrolyzed by PLE with high enantioselectivity. [11] These substrates have been used for the enantioselective construction of biologically relevant sugars (see Synthetic Applications below).

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Racemic mixtures of all of the substrates described above, as well as additional chiral diesters (such as the epoxy ester in equation (8)), may be resolved using PLE for kinetic resolution. [12] A significant disadvantage of kinetic resolution is a maximum yield of hydrolyzed product of 50%. However, if rapid racemization is occurring alongside hydrolysis (an example of dynamic kinetic resolution), a maximum yield of 100% is possible. [13]

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EsteraseScope6.png

Esterase enzymes may also be used for hydrolysis of base-sensitive monoesters. PLE has been applied to the synthesis of prostaglandins for the selective hydrolysis of the ester without destruction of the β-hydroxy ketone moiety. [14]

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Synthetic Applications

A number of synthetic targets possess hidden symmetry that may be discovered by applying a retrosynthetic "symmetrizing" transform. In the forward direction, this operation corresponds to a desymmetrization reaction. For instance, mevalonolactone may be synthesized rapidly from a symmetric diester via desymmetrizing hydrolysis, chemoselective reduction, and lactonization. [5] Although the product itself is asymmetric, desymmetrization and functional group manipulations permit its synthesis from an achiral starting material.

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EsteraseSynth1.png

Enantioselective hydrolysis of a conjugated diester followed by ozonolysis affords the skeleton of ribose. The resulting sugars are then carried on for the synthesis of nucleosides. [15]

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EsteraseSynth2.png

L-α-Methyldopa may be rapidly synthesized from an achiral malonate through a sequence beginning with desymmetrization. Subsequent chemoselective transformations convert the carboxylic acid to an amine. [16]

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EsteraseSynth3.png

Comparison with Other Methods

Other enzymes that may be used for asymmetric ester hydrolysis include electric eel acetylcholinesterase, [17] chymotrypsin, [3] and Baker's yeast. [18] The substrate scope of these enzymes differs from PLE, and in some cases they may provide hydrolyzed products in higher yield or enantioselectivity than PLE. Microorganisms may also be used for enantioselective hydrolysis; [19] however, difficulties associated with the handling of microorganisms have made these methods unpopular for organic synthesis.

Nonenzymatic methods for the differentiation of enantiotopic groups employ chiral catalysts or auxiliaries. For instance, the introduction of a chiral leaving group on both carboxylic acid groups of a meso diacid leads to selective attack by an achiral nucleophile at one of the (now) diastereotopic carbonyl groups. [20]

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Experimental Conditions and Procedure

Typical Conditions

Enzymatic reactions are limited by the need for aqueous solvent and near-neutral reaction conditions. PLE hydrolyses are typically carried out with a phosphate buffer to maintain the pH between 7 and 8. As solubility of the substrate in the aqueous medium is critical, a small amount of a polar organic co-solvent is sometimes added to the aqueous solution of the enzyme. Commercially available PLE is of sufficient purity for most applications.

Related Research Articles

Sharpless epoxidation chemical reaction

The Sharpless epoxidation reaction is an enantioselective chemical reaction to prepare 2,3-epoxyalcohols from primary and secondary allylic alcohols.

Sharpless asymmetric dihydroxylation is the chemical reaction of an alkene with osmium tetroxide in the presence of a chiral quinine ligand to form a vicinal diol. The reaction has been applied to alkenes of virtually every substitution, often high enantioselectivities are realized. Asymmetric dihydroxylation reactions are also highly site selective, providing products derived from reaction of the most electron-rich double bond in the substrate.

The Carroll rearrangement is a rearrangement reaction in organic chemistry and involves the transformation of a β-keto allyl ester into a α-allyl-β-ketocarboxylic acid. This organic reaction is accompanied by decarboxylation and the final product is a γ,δ-allylketone. The Carroll rearrangement is an adaptation of the Claisen rearrangement and effectively a decarboxylative Allylation.

Chiral auxiliary

A chiral auxiliary is a stereogenic group or unit that is temporarily incorporated into an organic compound in order to control the stereochemical outcome of the synthesis. The chirality present in the auxiliary can bias the stereoselectivity of one or more subsequent reactions. The auxiliary can then be typically recovered for future use.

<i>trans</i>-2-Phenyl-1-cyclohexanol chemical compound

trans-2-Phenyl-1-cyclohexanol is an organic compound. The two enantiomers of this compound are used in organic chemistry as chiral auxiliaries.

Petasis reaction

The Petasis reaction is the multi-component reaction of an amine, a carbonyl, and a vinyl- or aryl-boronic acid to form substituted amines.

An esterase is a hydrolase enzyme that splits esters into an acid and an alcohol in a chemical reaction with water called hydrolysis.

Asymmetric induction

Asymmetric induction in stereochemistry describes the preferential formation in a chemical reaction of one enantiomer or diastereoisomer over the other as a result of the influence of a chiral feature present in the substrate, reagent, catalyst or environment. Asymmetric induction is a key element in asymmetric synthesis.

In organic chemistry, kinetic resolution is a means of differentiating two enantiomers in a racemic mixture. In kinetic resolution, two enantiomers react with different reaction rates in a chemical reaction with a chiral catalyst or reagent, resulting in an enantioenriched sample of the less reactive enantiomer. As opposed to chiral resolution, kinetic resolution does not rely on different physical properties of diastereomeric products, but rather on the different chemical properties of the racemic starting materials. This enantiomeric excess (ee) of the unreacted starting material continually rises as more product is formed, reaching 100% just before full completion of the reaction. Kinetic resolution relies upon differences in reactivity between enantiomers or enantiomeric complexes. Kinetic resolution is a concept in organic chemistry and can be used for the preparation of chiral molecules in organic synthesis. Kinetic resolution reactions utilizing purely synthetic reagents and catalysts are much less common than the use of enzymatic kinetic resolution in application towards organic synthesis, although a number of useful synthetic techniques have been developed in the past 30 years.

Chiral derivatizing agent

A chiral derivatizing agent (CDA) also known as a chiral resolving reagent, is a chiral auxiliary used to convert a mixture of enantiomers into diastereomers in order to analyze the quantities of each enantiomer present within the mix. Analysis can be conducted by spectroscopy or by chromatography. The use of chiral derivatizing agents has declined with the popularization of chiral HPLC. Besides analysis, chiral derivatization is also used for chiral resolution, the actual physical separation of the enantiomers.

Desymmetrization in stereochemistry is the modification of a molecule that results in the loss of one or more symmetry elements. A common application of this class of reactions involves the introduction of chirality. Formally, such conversions required the loss of an improper axis of rotation. In other words, desymmetrisations convert prochiral precursors into chiral products.

Chiral Lewis acids (CLAs) are a type of Lewis acid catalyst that effects the chirality of the substrate as it reacts with it. In such reactions the synthesis favors the formation of a specific enantiomer or diastereomer. The method then is an enantioselective asymmetric synthesis reaction. Since they affect chirality, they produce optically active products from optically inactive or mixed starting materials. This type of preferential formation of one enantiomer or diastereomer over the other is formally known as an asymmetric induction. In this kind of Lewis acid. the electron-accepting atom is typically a metal, such as indium, zinc, lithium, aluminium, titanium, or boron. The chiral-altering ligands employed for synthesizing these acids most often have multiple Lewis basic sites that allow the formation of a ring structure involving the metal atom.

The [2,3]-Wittig rearrangement is the transformation of an allylic ether into a homoallylic alcohol via a concerted, pericyclic process. Because the reaction is concerted, it exhibits a high degree of stereocontrol, and can be employed early in a synthetic route to establish stereochemistry. The Wittig rearrangement requires strongly basic conditions, however, as a carbanion intermediate is essential. [1,2]-Wittig rearrangement is a competitive process.

Epoxidation with dioxiranes refers to the synthesis of epoxides from alkenes using three-membered cyclic peroxides, also known as dioxiranes.

Nucleophilic epoxidation is the formation of epoxides from electron-deficient double bonds through the action of nucleophilic oxidants. Nucleophilic epoxidation methods represent a viable alternative to electrophilic methods, many of which do not epoxidize electron-poor double bonds efficiently.

Reductions with hydrosilanes are chemical reactions that involve the combination of an organosilane (R3SiH) with an organic substrate containing unsaturated or electron-withdrawing functionality. Products in which the electron-withdrawing group has been replaced by hydrogen or the unsaturated group has been hydrogenated result.

Dynamic kinetic resolution in asymmetric synthesis

Dynamic kinetic resolution in chemistry is a type of kinetic resolution where 100% of a racemic compound can be converted into a enantiopure compound. It is applied in asymmetric synthesis. Asymmetric synthesis has become a much explored field due to the challenge of creating a compound with a single 3D structure. Even more challenging is the ability to take a racemic mixture and have only one chiral product left after a reaction. One method that has become an exceedingly useful tool is dynamic kinetic resolution (DKR). DKR utilizes a center of a particular molecule that can be easily epimerized so that the (R) and (S) enantiomers can interconvert throughout the reaction process. At this point the catalyst can selectively lower the transition state energy of a single enantiomer, leading to almost 100% yield of one reaction pathway over the other. The figure below is an example of an energy diagram for a compound with an (R) and (S) isomer.

Shiina esterification is an organic chemical reaction that synthesizes carboxylic esters from nearly equal amounts of carboxylic acids and alcohols by using aromatic carboxylic acid anhydrides as dehydration condensation agents. In 1994, Prof. Isamu Shiina reported an acidic coupling method using Lewis acid, and, in 2002, a basic esterification using nucleophilic catalyst.

Phosphoramidite ligand

A phosphoramidite ligand is a chiral monodentate phosphine ligand, widely used for enantioselective synthesis. They were invented by Dutch chemist Ben Feringa. The introduction of phosphoramidite ligands challenged the notion that high flexibility in the metal–ligand complex is detrimental for high stereo control.

References

  1. ↑ Ohno, M.; Otsuka, M. Org. React. 1989, 37, 1. doi : 10.1002/0471264180.or037.01
  2. ↑ Rétey, J.; Robinson, J. Stereospecificity in Organic Chemistry and Enzymology, Verlag Chemie, Weinheim, 1982.
  3. 1 2 Cohen, S.; Khedouri, E. J. Am. Chem. Soc.1961, 83, 1093.
  4. ↑ Zemlicka, J.; Craine, L.; Heeg, M.-J.; Oliver, J. J. Org. Chem.1988, 53, 937.
  5. 1 2 Huang, F.-C.; Lee, L. F. H.; Mittal, R. S. D.; Ravikumar, P. R.; Chan, J. A.; Sih, C. J.; Capsi, E.; Eck, C. R. J. Am. Chem. Soc.1975, 97, 4144.
  6. ↑ Lam, L. K. P.; Hui, R. A. H. F.; Jones, J. B. J. Org. Chem.1986, 51, 2047.
  7. ↑ Björkling, F.; Boutelje, J.; Gatenbeck, S.; Hult, K.; Norin, T.; Szmulik, P. Tetrahedron1985, 41, 1347.
  8. ↑ Mohr, P.; Waespe-Sarevi, N.; Tamm, C.; Gawronska, K.; Gawronski, J. Helv. Chim. Acta1983, 66, 2501.
  9. ↑ Sabbioni, G.; Shea, M. L.; Jones, J. B. J. Chem. Soc., Chem. Commun.1984, 236.
  10. ↑ Schneider, M.; Engel, N.; Hönicke, P.; Heinemann, G.; Görisch, H. Angew. Chem. Int. Ed. Engl.1984, 23, 67.
  11. ↑ Guanti, G.; Banfi, L.; Narisano, E.; Riva, R.; Thea, S. Tetrahedron Lett.1986, 27, 4639.
  12. ↑ Mohr, P.; Rösslein, L.; Tamm, C. Helv. Chim. Acta1987, 70, 142.
  13. ↑ Allen, J.; Williams, J. Tetrahedron Lett.1996, 37, 1859.
  14. ↑ Hazato, A.; Tanaka, T.; Toru, T.; Okamura, N.; Bannai, K.; Sugiura, S.; Manabe, K.; Kurozumi, S. Nippon Kagaku Kaishi1983, 9, 1390 [C.A., 100, 120720q (1984)].
  15. ↑ Ohno, M.; Kobayashi, S.; Adachi, K. in Enzymes as Catalysts in Organic Synthesis, Schneider, M. P. Ed., D. Reidel Publishing, Dordrecht, 1986, pp. 123–142.
  16. ↑ Björkling, F.; Boutelje, J.; Gatenbeck, S.; Hult, K.; Norin, T. Tetrahedron Lett.1985, 26, 4957.
  17. ↑ Deardorff, D. R.; Matthews, A. J.; McMeekin, D. S.; Craney, C. L. Tetrahedron Lett.1986, 27, 1255.
  18. ↑ Kerscher, V.; Kreiser, W. Tetrahedron Lett.1987, 28, 531.
  19. ↑ Kotani, H.; Kuze, Y.; Uchida, S.; Miyabe, T.; Iimori, T.; Okano, K.; Kobayashi, S.; Ohno, M.; Agric. Biol. Chem.1983, 47, 1363.
  20. ↑ Nagao, Y.; Ikeda, T.; Yagi, M.; Fujita, E.; Shiro, M. J. Am. Chem. Soc.1982, 104, 2079.
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